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Velocity-difference induced focusing (V-DIF) with a dynamic pH junction in capillary electrophoresis (CE) using a sample with a pH different from that of the background electrolyte (BGE) was developed in our group, but the mechanism was not well understood. In this work, the mechanism of this focusing technique was first studied using an appropriate dye to monitor the pH of the BGE and the sample during the focusing process. A mechanism was proposed based on the experimental results. This technique was then applied to serotonin to improve the detection limit when CE was used with a UV absorption detector. It was also applied to focus amino acids, peptides, and proteins to improve the concentration sensitivity. It is found that the pKa rather than the pI of the analytes is the key criterion for selecting the pH for the sample and for the BGE to obtain the optimum focusing for these molecules. Since UV detection only provides migration time information, more structure information is obtained by using a photodiode array (PDA) and mass spectrometer (MS) for peak identification. Comparisons were made between the PDA detection and MS detection for aromatic amino acids with V-DIF using a dynamic pH junction. This V-DIF technique was then applied to non-aromatic amino acids with MS detection. It was used at low pH with positive ESI-MS detection and at high pH with negative ESI-MS ionization. The results of the two methods were compared and discussed. Finally, the preparative operation of continuous flow counterbalanced CE (FCCE) was studied. The effects of larger sample volumes and multiple capillary systems on improving the purification yield were investigated.